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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Translational Medicine 1/2017

Proteomic analysis of protein purified derivative of Mycobacterium bovis

Journal of Translational Medicine > Ausgabe 1/2017
Sante Roperto, Mariaconcetta Varano, Valeria Russo, Roberta Lucà, Monica Cagiola, Marco Gaspari, Dora Maria Ceccarelli, Giovanni Cuda, Franco Roperto
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Electronic supplementary material

The online version of this article (doi:10.​1186/​s12967-017-1172-1) contains supplementary material, which is available to authorized users.



Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB).


Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system.


Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920.


This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.
Additional file 1: Table S1. This table shows: the list of the identified proteins within all four bPPD preparations, ordered locus names of M. bovis (Mb) and M. tuberculosis, strain H37Rv, (Rv), functional categories of identified proteins, their molecular mass, gene and protein lengths and gene name and protein function. A comparative protein expression profile with two previous bPPD proteomic analysis is also shown. Differences in functional category among identified proteins are in blue; differences in molecular mass, gene and protein lengths are in red. These findings were from TubercuList, BoviList, Uniprot and KEGG databases (See text for further details).
Additional file 2: Table S2. The list of identified proteins in bPPDs. Sequence coverage, number of unique peptides, as well as total weighted spectra as a semi-quantitative measure of protein abundance, are reported.
Additional file 3: Table S3. Dimethyl labeling-based quantification of proteins present in samples I t, NLand I p relative to sample S. Result of single replicates as well as average H:L and M:L ratios, their associated p-values and number of observations (unique peptides) are reported.
Additional file 4: Table S4. Proteins found differentially abundant in samples I t, NL and I p compared to sample S. The file contains three data sheets reporting 44, 34 and 30 proteins found at significantly different levels in samples It, NL and Ip, respectively. These proteins were mostly present at very low levels in all preparations. Fold changes >2 and <0.5 are reported in two separate lists.
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